Hosuk:LabNotes/2013-11-19: Difference between revisions
Jump to navigation
Jump to search
>Hosuki78 No edit summary |
>Hosuki78 No edit summary |
||
(10 intermediate revisions by the same user not shown) | |||
Line 8: | Line 8: | ||
====Procedure==== | ====Procedure==== | ||
*2048 x | *20x obj, 2048 x 2048 resolution, 16bit, | ||
*Scan Freq. 600Hz --> Image quality is not different from 400Hz. | |||
*Sample : MatTek Dish, made at 07/09 | *Sample : MatTek Dish, made at 07/09 | ||
*Each stripping, and annealing : 15min incubation @ RT | *Each stripping, and annealing : 15min incubation @ RT | ||
*Each time the concentration of the dye-labeled probe is 2uM, | |||
*The volume added is 100uL. | |||
*Process Steps | *Process Steps | ||
* | *#Step 0 : Strip | ||
* | *#Step 1 : Cy5 in 2x SSC | ||
* | *#Step 2 : Strip | ||
* | *#Step 3 : Cy5 in 70% Formamide + 2x SSC | ||
* | *#Step 4 : Strip | ||
* | *#Step 5 : Cy5 in 10% Formamide + 2x SSC | ||
* | *#Step 6 : Strip | ||
* | *#Step 7 : Cy5 in 30 % Formamide + 2x SSC | ||
* | *#Step 8 : Strip | ||
* | *#Step 9 : Cy5 in 50% Formamide + 2x SSC | ||
* | *#Step 10 : Strip | ||
* | *#Step 11 : Cy5 in 70% Formamide + 2x SSC | ||
Line 34: | Line 37: | ||
=====1st Rolony Count===== | |||
*Before Step 0 (At the beginning Cy3 probe were annealed) --> 5932 | |||
*Step 1 : Cy5 in 2x SSC --> 5469 | |||
*Step 3 : Cy5 in 70% Formamide + 2x SSC --> 543 | |||
*Step 5 : Cy5 in 10% Formamide + 2x SSC --> 5413 | |||
*Step 7 : Cy5 in 30 % Formamide + 2x SSC --> 5446 | |||
*Step 9 : Cy5 in 50% Formamide + 2x SSC --> 4793 | |||
*Step 11 : Cy5 in 70% Formamide + 2x SSC --> 421 | |||
Line 63: | Line 65: | ||
=====Number of 1st Rolony vs. Formamide %===== | =====Number of 1st Rolony vs. Formamide %===== | ||
[[File:Chart_DyeProbeTest_Cy5-1stRolony_Step1-3-5-7-9-11.png| | [[File:Chart_DyeProbeTest_Cy5-1stRolony_Step1-3-5-7-9-11.png|850px]] | ||
=====1st Rolonies along each step===== | =====1st Rolonies along each step===== | ||
[[File:DyeProbeTest_Cy5-1stRolony_Step1-3-5-7-9-11_label.png| | [[File:DyeProbeTest_Cy5-1stRolony_Step1-3-5-7-9-11_label.png|850px]] | ||
*'''Raw images''' | |||
**[[Media:DyeProbeTest_MatTek20130709_1stRolony_Cy3_2k_600Hz_ch00_MIP.tif|Before Step0 (Cy3, ]][[Media:DyeProbeTest_MatTek20130709_1stRolony_Cy3_2k_600Hz_ch01_MIP.tif|BF)]], | |||
**[[Media:DyeProbeTest.lif_MatTek20130709_Step1_Cy5_ch00_MIP.tif|Step1 (Cy5]], [[Media:DyeProbeTest.lif_MatTek20130709_Step1_Cy5_ch01_MIP.tif|BF)]], [[Media:DyeProbeTest.lif_MatTek20130709_Step3_Cy5-Formamide70_Cy5_ch00_MIP.tif|Step3]], [[Media:DyeProbeTest.lif_MatTek20130709_Step5_Cy5-Formamide10_Cy5_ch00_MIP.tif|Step5]], [[Media:DyeProbeTest.lif_MatTek20130709_Step7_Cy5-Formamide30_Cy5_ch00_MIP.tif|Step7]], [[Media:DyeProbeTest.lif_MatTek20130709_Step9_Cy5-Formamide50_Cy5_ch00_MIP.tif|Step9 (Cy5, ]][[Media:DyeProbeTest.lif_MatTek20130709_Step9_Cy5-Formamide50_Cy5_ch01_MIP.tif|BF)]], [[Media:DyeProbeTest.lif_MatTek20130709_Step11_Cy5-Formamide70_Cy5_ch00_MIP.tif|Step11]] | |||
*'''Stage was moveed a little from Step9 by mistake, so the location was re-configured.''' | |||
===Dye labeled Probe Annealing Test – with dcProbe3-Cy3 : Non-specific binding=== | |||
*Figure out lower limit by Formamide concentration : to minimize non-specific binding | |||
*dcProbe3-Cy3 does not have target in rolony, so we can see how many spots due to the non-specific binding. | |||
*Done @ 11/19 | |||
====Procedure==== | |||
*20x obj, 2048 x 2048 resolution, 16bit, | |||
*Scan Freq. 600Hz --> Image quality is not different from 400Hz. | |||
*Sample : MatTek Dish, made at 07/09 | |||
*Each stripping, and annealing : 15min incubation @ RT | |||
*Each time the concentration of the dye-labeled probe is 1uM, | |||
*The volume added is 100uL. | |||
*Process Steps | |||
Process Step | |||
Step 0 : Strip | |||
Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC 160 | |||
Step 2 : Strip ~34 | |||
Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC 67 | |||
Step 4 : Strip | |||
Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC 66 | |||
*#Step 0 : Strip | |||
*#Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC | |||
*#Step 2 : Strip | |||
*#Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC | |||
*#Step 4 : Strip | |||
*#Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC | |||
====Result==== | |||
*30% seemed optimal concentration for less non-specific binding as well as for target-specific binding.. | |||
*Need to get 3 data point to confirm those results (for the future when I set up an automation system. | |||
=====Spot Count===== | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Formamide (%)''' | |||
| align="center" style="background:#f0f0f0;"|'''Spot Count''' | |||
|- | |||
| align="center" | Strip || align="center" | 34 | |||
|- | |||
| align="center" | 0 || align="center" | 160 | |||
|- | |||
| align="center" | 30 || align="center" | 67 | |||
|- | |||
| align="center" | 40 || align="center" | 66 | |||
|} | |||
[[File:Chart_DyeProbeTest_dcProbe3_Step1-2-3-5.png|800px]] | |||
*'''Raw images''' | |||
**[[Media:DyeProbeTest.lif_MatTek20130709_Step1_dcProbe3_Cy3_ch00_MIP.tif|Step1 (Cy3, ]][[Media:DyeProbeTest.lif_MatTek20130709_Step1_dcProbe3_Cy3_ch01_MIP.tif|BF)]], [[Media:DyeProbeTest.lif_MatTek20130709_Step2_Strip_Cy3_ch00_MIP.tif|Step2]], [[Media:DyeProbeTest.lif_MatTek20130709_Step3_dcProbe3_Formamide30_Cy3_ch00_MIP.tif|Step3]], [[Media:DyeProbeTest.lif_MatTek20130709_Step4_Strip_Cy3_ch00_MIP.tif|Step4]], [[Media:DyeProbeTest.lif_MatTek20130709_Step5_dcProbe3_Formamide40_Cy3_ch00_MIP.tif|Step5]] |
Latest revision as of 05:37, 20 November 2013
Dye labeled Probe Annealing Test – with Cy3-1st Rolony[edit]
- Figure out upper limit by Formamide concentration
- Done @ 11/18
Procedure[edit]
- 20x obj, 2048 x 2048 resolution, 16bit,
- Scan Freq. 600Hz --> Image quality is not different from 400Hz.
- Sample : MatTek Dish, made at 07/09
- Each stripping, and annealing : 15min incubation @ RT
- Each time the concentration of the dye-labeled probe is 2uM,
- The volume added is 100uL.
- Process Steps
- Step 0 : Strip
- Step 1 : Cy5 in 2x SSC
- Step 2 : Strip
- Step 3 : Cy5 in 70% Formamide + 2x SSC
- Step 4 : Strip
- Step 5 : Cy5 in 10% Formamide + 2x SSC
- Step 6 : Strip
- Step 7 : Cy5 in 30 % Formamide + 2x SSC
- Step 8 : Strip
- Step 9 : Cy5 in 50% Formamide + 2x SSC
- Step 10 : Strip
- Step 11 : Cy5 in 70% Formamide + 2x SSC
Result[edit]
- 0~30% Formamide concentration showed similar number of rolony, but the number decreased at 70%.
- Need to try 20%, 30%, 40%, twice at each time in order to confirm 30% is maximum condition!
1st Rolony Count[edit]
- Before Step 0 (At the beginning Cy3 probe were annealed) --> 5932
- Step 1 : Cy5 in 2x SSC --> 5469
- Step 3 : Cy5 in 70% Formamide + 2x SSC --> 543
- Step 5 : Cy5 in 10% Formamide + 2x SSC --> 5413
- Step 7 : Cy5 in 30 % Formamide + 2x SSC --> 5446
- Step 9 : Cy5 in 50% Formamide + 2x SSC --> 4793
- Step 11 : Cy5 in 70% Formamide + 2x SSC --> 421
Formamide (%) | 1st Rolony Count |
0 | 5469 |
10 | 5413 |
30 | 5446 |
50 | 4793 |
70 | 482 |
- 0% : average from 2 results
Number of 1st Rolony vs. Formamide %[edit]
File:Chart DyeProbeTest Cy5-1stRolony Step1-3-5-7-9-11.png
1st Rolonies along each step[edit]
File:DyeProbeTest Cy5-1stRolony Step1-3-5-7-9-11 label.png
- Raw images
- Stage was moveed a little from Step9 by mistake, so the location was re-configured.
Dye labeled Probe Annealing Test – with dcProbe3-Cy3 : Non-specific binding[edit]
- Figure out lower limit by Formamide concentration : to minimize non-specific binding
- dcProbe3-Cy3 does not have target in rolony, so we can see how many spots due to the non-specific binding.
- Done @ 11/19
Procedure[edit]
- 20x obj, 2048 x 2048 resolution, 16bit,
- Scan Freq. 600Hz --> Image quality is not different from 400Hz.
- Sample : MatTek Dish, made at 07/09
- Each stripping, and annealing : 15min incubation @ RT
- Each time the concentration of the dye-labeled probe is 1uM,
- The volume added is 100uL.
- Process Steps
Process Step Step 0 : Strip Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC 160 Step 2 : Strip ~34 Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC 67 Step 4 : Strip Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC 66
- Step 0 : Strip
- Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC
- Step 2 : Strip
- Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC
- Step 4 : Strip
- Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC
Result[edit]
- 30% seemed optimal concentration for less non-specific binding as well as for target-specific binding..
- Need to get 3 data point to confirm those results (for the future when I set up an automation system.
Spot Count[edit]
Formamide (%) | Spot Count |
Strip | 34 |
0 | 160 |
30 | 67 |
40 | 66 |
File:Chart DyeProbeTest dcProbe3 Step1-2-3-5.png